Contribution of APOBEC3G/F activity to the development of low-abundance drug-resistant human immunodeficiency virus type 1 variants.

Plasma drug-resistant minority human immunodeficiency virus type 1 variants (DRMVs) increase the risk of virological failure to first-line non-nucleoside reverse transcriptase inhibitor antiretroviral therapy (ART). The origin of DRMVs in ART-naive patients, however, remains unclear. In a large pan-European case-control study investigating the clinical relevance of pre-existing DRMVs using 454 pyrosequencing, the six most prevalent plasma DRMVs detected corresponded to G-to-A nucleotide mutations (V90I, V106I, V108I, E138K, M184I and M230I). Here, we evaluated if such DRMVs could have emerged from apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3G/F (APOBEC3G/F) activity. Out of 236 ART-naive subjects evaluated, APOBEC3G/F hypermutation signatures were detected in plasma viruses of 14 (5.9%) individuals. Samples with minority E138K, M184I, and M230I mutations, but not those with V90I, V106I or V108I, were significantly associated with APOBEC3G/F activity (Fisher's P < 0.005), defined as the presence of > 0.5% of sample sequences with an APOBEC3G/F signature. Mutations E138K, M184I and M230I co-occurred in the same sequence as APOBEC3G/F signatures in 3/9 (33%), 5/11 (45%) and 4/8 (50%) of samples, respectively; such linkage was not found for V90I, V106I or V108I. In-frame STOP codons were observed in 1.5% of all clonal sequences; 14.8% of them co-occurred with APOBEC3G/F signatures. APOBEC3G/F-associated E138K, M184I and M230I appeared within clonal sequences containing in-frame STOP codons in 2/3 (66%), 5/5 (100%) and 4/4 (100%) of the samples. In a re-analysis of the parent case control study, the presence of APOBEC3G/F signatures was not associated with virological failure. In conclusion, the contribution of APOBEC3G/F editing to the development of DRMVs is very limited and does not affect the efficacy of non-nucleoside reverse transcriptase inhibitor ART.

Human immunodeficiency virus (HIV) antibody avidity testing to identify recent infection in newly diagnosed HIV type 1 (HIV-1)-seropositive persons infected with diverse HIV-1 subtypes.

A guanidine-based antibody avidity assay for the identification of recently acquired human immunodeficiency virus type 1 (HIV-1) infection was evaluated. The kinetics of maturation of antibody avidity were determined prospectively in 23 persons undergoing acute seroconversion followed for up to 1,075 days. Avidity indices (AI) of

Comparative evaluation of the performance of the Abbott real-time human immunodeficiency virus type 1 (HIV-1) assay for measurement of HIV-1 plasma viral load following automated specimen preparation.

The performance of the new Abbott real-time human immunodeficiency virus type 1 (HIV-1) assay for HIV-1 RNA load determination in plasma was compared to that of the Abbott LCx HIV-1 RNA quantitative assay following automated RNA isolation by the Abbott m1000 extractor. The measured viral loads of 89 clinical specimens differed by mean 0.19 log10 copies/ml (95% confidence interval, 0.12 to 0.26 log10 copies/ml). Although the difference in viral load determinations was positively skewed in favor of the LCx assay, it did not reach statistical significance (P = 0.42). Results were linearly associated (R2 = 0.94) and strongly correlated (R = 0.96). Good performance was observed with HIV-1 subtypes other than B and circulating recombinant forms, although results obtained with two subtype G specimens and one H specimen showed a more substantial difference.

Differences in dietary patterns of nonsmoking adults married to smokers vs. nonsmokers.

PURPOSE: To compare dietary intakes of nonsmoking adults married to smokers or nonsmokers. DESIGN: Respondents to the U.S. Department of Agriculture's Continuing Survey of Food Intakes by Individuals (CSFII), 1994 to 1996 (response rate = 76.1% for 2 days of dietary intake). Nonsmoking adults aged 18 and older were grouped according to the smoking status of their spouse. SETTING: In-home interviews in all 50 states and Washington, D.C. SUBJECTS: The selected sample included 757 men and 754 women who were married to nonsmokers, and 197 men and 262 women who were married to smokers. MEASURES: Selected demographic variables, food group servings, food energy, and densities of selected nutrients were compared using chi 2 and analysis of covariance. RESULTS: Men and women married to smokers had greater (p < or = .025) energy-adjusted intakes of total and saturated fat but significantly lower (p < or = .05) energy-adjusted intakes of fiber and vitamin A. Men married to smokers consumed significantly more (p < .025) energy-adjusted cholesterol and ethanol but significantly less calcium (p = .026); women married to smokers consumed significantly less (p = .014) energy-adjusted folate. Men married to smokers consumed significantly more (p < or = .05) alcoholic beverages, coffee, and soft drinks; women married to smokers consumed significantly less water (p = .014) but more cheese and table sweeteners (p < or = .05). CONCLUSIONS: Nonsmoking men and women who were married to smokers had compromised dietary intakes. Nonsmoking men whose wives smoked, in particular, had unhealthy diets due to elevated intakes of fat and cholesterol and low intakes of vitamin A, calcium, and fiber. Health professionals should continue to provide tobacco cessation instruction and dietary guidance, but also be aware of at-risk patients' immediate family members who likely share an increased risk of disease because of poor diet quality and exposure to environmental tobacco smoke.

P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes.

Recently, sandwich-cultured (SC) rat hepatocytes have been used as an in vitro model to assess biliary excretion of drugs and xenobiotics. The purpose of the present study was to validate the use of SC rat hepatocytes for the in vitro assessment of P-glycoprotein (P-gp)-mediated biliary drug excretion. The specific and fluorescent P-gp substrate rhodamine 123 (Rh123) and the P-gp substrate digoxin were selected as model compounds. Rh123 and digoxin accumulation and Rh123 efflux under standard and Ca(2+)-free conditions were quantified in SC rat hepatocytes to determine substrate secretion into canalicular networks in vitro. The major role of P-gp in the biliary excretion of these compounds was confirmed by inhibition experiments with the potent P-gp inhibitor GF120918. Hepatocyte culture conditions, including media type and time in culture, significantly affected Rh123 biliary excretion. P-gp expression, as assessed by Western blot, was increased with culture time. Dexamethasone (an in vivo inducer of P-gp) concentrations ranging from 0.01 to 1 microM in the cell culture medium did not influence P-gp expression or Rh123 biliary excretion. Rh123 and digoxin biliary clearance values, predicted from SC rat hepatocyte data, were consistent with values reported in vivo and in isolated perfused rat liver studies. In conclusion, the results of this study demonstrate the utility of SC rat hepatocytes as an in vitro model to study and predict the biliary excretion of P-gp substrates.

Mannose-binding lectin accelerates complement activation and increases serum killing of Neisseria meningitidis serogroup C.

The capacity for different lipo-oligosaccharide (LOS) sialylation patterns of Neisseria meningitidis serogroup C to influence the binding and function of the innate humoral component, mannose-binding lectin (MBL), was investigated. By use of flow cytometry and immunogold electron microscopy, a clinical isolate with reduced endogenous LOS sialylation was found to bind more MBL than did strains with higher endogenous sialylation. MBL binding was reduced but not ablated if the same strain was allowed to exogenously sialylate its LOS structures after incubation with cytidine-5'-monophospho-neuraminic acid. MBL binding led to an increased rate of complement activation, with enhanced deposition of the complement components C4 and C5b-9, and this correlated with an increase in bactericidal activity. LOS sialylation appears to be an important determinant of MBL binding to N. meningitidis and can modulate complement-dependent killing of the bacterium. These findings could explain the observed susceptibility to meningococcal disease of individuals genetically deficient in MBL.

Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein.

Human P-glycoprotein, the MDR1 gene product, requires both Mg(2+)-ATP binding and hydrolysis to function as a drug transporter; however, the mechanism(s) defining these events is not understood. In the present study, we explored the nature of Mg(2+)-ATP binding in the N-terminal nucleotide-binding domain of human P-glycoprotein and identified the minimal functional unit required for specific ATP binding. Recombinant proteins encompassing amino acids within the region beginning at 348 and ending at 707 were expressed in Escherichia coli, purified from inclusion bodies under denaturing conditions, and renatured by rapid dilution. The ability of ATP to interact with these proteins was examined by use of the photoactive ATP analogue [alpha-(32)P]-8-azido-ATP. Photoaffinity labeling of recombinant proteins identified the region between amino acids 375 and 635 as the region necessary to obtain specific ATP-binding properties. Specific protein labeling was saturable, enhanced by Mg(2+), and inhibited by ATP. Recombinant proteins confined within the region beginning at amino acid 392 and ending at amino acid 590 demonstrated nonspecific [alpha-(32)P]-8-azido-ATP labeling. Nonspecific labeling was not enhanced by Mg(2+) and was inhibited only by high concentrations of ATP. Using a D555N mutated protein, we found that the conserved aspartate residue in the Walker B motif plays a role in magnesium-enhanced ATP-binding. Taken together, these data define the region of the N-terminal nucleotide-binding domain of P-glycoprotein that is required for specific ATP binding and suggest that magnesium may play a role in stabilizing the ATP-binding site.

Child care and employment in relation to infants' disabilities and risk factors.

The impact of disability- and risk-related characteristics of 166 infants on their mothers' employment and child-care characteristics and decisions was investigated. Mothers' employment plans and child-care decisions were affected by their children's special needs (chronic health problems; use of adaptive equipment; total risks; diagnosis; and mental, motor, and adaptive functioning). Child-care characteristics were related most consistently to the children's diagnoses. Infants diagnosed with Down syndrome compared with those whose developmental delays were of unknown origin entered child care earlier and were more likely to be in centers and to be receiving lower quality care.

Effect of multidrug resistance modulators on the hepatobiliary disposition of doxorubicin in the isolated perfused rat liver.

P-Glycoprotein (P-gp)-mediated multidrug resistance (MDR) in cancer cells may be modulated by competitive inhibitors of P-gp. In the liver, P-gp is localized on the canalicular membrane of hepatocytes. Quinidine and GF120918 inhibit the transport of P-gp substrates, including doxorubicin. Competitive inhibition of P-gp transport may alter biliary excretion of substrates. This study was designed to examine the effects of MDR modulators on the hepatobiliary disposition of doxorubicin and to elucidate the site(s) of drug-modulator interaction using pharmacokinetic modeling techniques. Livers from male Sprague Dawley rats were isolated and perfused for 2 h at 37 degrees C with recirculating male rat blood. MDR modulator (16.8-480 microg of GF120918 or 0.3-3.0 mg of quinidine) or vehicle (buffer or DMSO, respectively) was administered as a bolus to the perfusate reservoir 5 min prior to the addition of doxorubicin (464 microg). Perfusate and bile were collected during the perfusion, the liver was homogenized after the perfusion, and samples were analyzed by high-pressure liquid chromatography for doxorubicin and the major metabolite doxorubicinol. In the presence of GF120918, the biliary excretion of doxorubicin and doxorubicinol was decreased significantly without alterations in doxorubicin perfusate concentrations or doxorubicin and doxorubicinol liver concentrations. In the presence of quinidine, the biliary excretion of doxorubicin was reduced significantly; however, doxorubicinol recovery in bile was not altered. The perfusate and liver concentrations of doxorubicin were not altered by quinidine; doxorubicinol liver concentrations were increased. A series of pharmacokinetic models were evaluated incorporating perfusate, liver, and bile compartments to describe the disposition of doxorubicin and doxorubicinol in the isolated perfused rat liver. The model that best described these data, based on goodness-of-fit criteria, included first-order rate constants for all disposition processes. On the basis of this model, the rate-limiting process for doxorubicin and doxorubicinol elimination was biliary excretion. In the presence of GF120918, rate constants associated with doxorubicin and doxorubicinol canalicular egress were decreased, and other doxorubicinol disposition pathways were increased slightly. Quinidine was associated with a decrease in doxorubicin canalicular egress, doxorubicinol formation, and other doxorubicinol pathways. Pharmacokinetic modeling of the data supported the hypothesis that decreased biliary excretion of doxorubicin in the isolated perfused rat liver, as determined by mass-balance analysis, was due to interactions at the canalicular membrane. The present study further supports the utility of pharmacokinetic modeling in identifying sites of drug interactions within the hepatobiliary system. This approach may be particularly useful in predicting the effects of perturbations in hepatic translocation processes on the hepatobiliary disposition of drugs and derived metabolites.

Perceptions of emotional support from mother and friend in middle childhood: links with social-emotional adaptation and preschool attachment security.

Children's (N = 58) perceptions of emotional support from mother and best friend were assessed at age 8. Perceptions of support from mother were predicted by attachment security at age 4, suggesting continuity in the children's internal working model of self in relation to mother. Preschool attachment security predicted age 8 perceptions of maternal support better than the mother's actual behavior at age 8. Identification of the best friend as a member of one's emotional support network was not related to security, but was positively related to social competence. However, among insecurely attached children, the greater the reliance on the best friend for emotional support, the greater the externalizing problems. Compensatory effects of best friend support on the social-emotional adaptation of insecurely attached children were not found.

Structure-based design of substituted diphenyl sulfones and sulfoxides as lipophilic inhibitors of thymidylate synthase.

Six new diphenyl sulfoxide and five new diphenyl sulfones were designed, synthesized, and tested for their inhibition of human and Escherichia coli thymidylate synthase (TS) and of the growth of cells in tissue culture. The best sulfoxide inhibitor of human TS was 3-chloro-N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4- (phenylsulfinyl)-N-(prop-2-ynyl)-aniline (7c) that had a Ki of 27 nM. No sulfone improved on TS inhibition by the previously reported 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2- ynylamino)phenyl phenyl sulfone (Ki = 12 nM). Nevertheless, one sulfone, 4-((2-chlorophenyl)sulfonyl)-N-((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)-N-(prop-2-ynyl)aniline, was selected, on the basis of its inhibition of both TS and cell growth, for antitumor testing; it gave a 61% increase in life span to mice bearing the thymidino kinase-deficient L5178Y (TK-) lymphoma. A crystal structure of N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4-((2- methylphenyl)sulfinyl)-N-(prop-2-ynyl)aniline complexed with E. coli TS was solved and revealed selective binding of one sulfoxide enantiomer. AMBER calculations showed that the enantioselection was due to asymmetric electrostatic effects at the mouth of the active site. In contrast, a similar crystal structure of the sulfoxide 7c, along with AMBER calculations, indicated that both enantiomers bound, but with different affinities. The side chain of Phe176 shifted in order to structurally accommodate the chlorine of the more weakly bound enantiomer.

Erythrocyte morphology and filterability in rats fed on diets containing different fats and oils.

The present study aimed to determine if the type of dietary fat or oil affects erythrocyte morphology and/or filterability in normal healthy rats. A feeding trial was carried out, in which nine groups of nine rats were fed on diets containing one of the following treatments (test fats or oils): anhydrous milk fat, anhydrous milk fat after passage through a column of active carbon, palm oil, MaxEPA fish oil, hydrogenated coconut oil, anhydrous tallow shortening, margarine hardstock, olive oil and soyabean oil. The test fats or oils supplemented with 10 g safflower-seed oil/kg were incorporated into otherwise nutritionally adequate diets so that the test fat or oil plus safflower-seed oil contributed 35% of the gross energy of the diet. The rats were fed for 10 weeks. Diet had a significant effect on five of the six classes of erythrocytes identified, and the proportion of cells in each class was shown to be dependent on diet. However, the attribute causing the dependence was not clear. There was no significant effect of diet on erythrocyte filterability index. There was no statistical correlation between erythrocyte filterability index and morphology. Although it has been observed that diet, particularly fish oil, can improve the filterability of erythrocytes once filterability is impaired, the effect of diet on erythrocyte filterability in normal healthy animals including humans is unclear. The importance of the differences in erythrocyte morphology due to diet is also unclear. Both areas deserve further investigation.

Hepatobiliary disposition of valproic acid and valproate glucuronide: use of a pharmacokinetic model to examine the rate-limiting steps and potential sites of drug interactions.

Previous work in this laboratory has suggested that the nonlinear disposition of valproic acid (VPA) in the rat may be due to nonlinear distribution of VPA into the liver. The present study was undertaken to elucidate further the hepatobiliary disposition of VPA. VPA (0.1-2 mmol/L) was incubated with isolated rat hepatocytes in vitro. Uptake of [(3)H]-VPA was linear from 10 to 50 seconds, with minimal (<7 percent) biotransformation. The initial velocity of VPA uptake varied in proportion with the extracellular concentration and was temperature independent, suggesting that VPA traverses the hepatocyte membrane predominantly by passive diffusion. In separate studies, the hepatobiliary disposition of VPA (20mg) was examined in the isolated perfused rat liver (IPL). A pharmacokinetic model was developed to describe the influence of phenobarbital on the hepatobiliary disposition of VPA and valproate glucuronide (V-G) in the IPL; all processes governing VPA and V-G disposition appeared to be linear. Acute administration of phenobarbital to the liver (1.12 mg) decreased the rate constant for canalicular egress of V-G (0.0489 +/- 0.0266 vs. 0.164 +/- 0.075 min(-1)). In vivo pretreatment with phenobarbital (75 mg/kg/d x 5 d) before liver isolation decreased the biliary excretion of both VPA (1.06E-04 +/- 0.27E-04 vs. 2.76E-04 +/- 0.45E-04 min(-1)) and V-G (5.63E- 03 +/- 1.98E-03 vs. 1.74E-02 +/- 0.5E-02 min(-1)), and increased the apparent volume of distribution of VPA (84.6 +/- 2.2 vs. 72.3 +/- 2.1 mL). In vivo phenobarbital pretreatment a changed V-G excretion from a formation to an elimination rate-limited process. These results are consistent with phenobarbital-associated impairment of canalicular egress of some organic anions. This work further supports the utility of pharmacokinetic modeling in: (1) determining the rate-limiting steps in hepatobiliary drug disposition and (2) identifying sites of drug interactions within the hepatobiliary system that may not be evident based on conventional mass-balance analysis.

Combination tissue plasminogen activator and ticlopidine therapy in a rabbit model of acute thromboembolic stroke.

The successful application of thrombolytic therapy to acute myocardial infarction has prompted a reinvestigation of thrombolytic therapy for acute stroke. However, an examination of safety and efficacy of thrombolytic therapy in acute thromboembolic stroke has precluded the entry of patients taking either antiplatelet or anticoagulant therapy. It was therefore of interest, in an established rabbit model of thromboembolic stroke, to examine the use of tissue plasminogen activator therapy in combination with ticlopidine treatment. Following ticlopidine administration (10 mg kg-1, i.v., daily for 5 days), rabbits (n = 7) were embolized by injecting a tin-laden clot into the internal carotid artery with clot placement confirmed by x-ray. Three hours later, t-PA was initiated as a square-wave pulse (6.3 mg kg-1 total dose, given as a 20% bolus, with the remainder administered over 2 h as a continuous infusion). The protocol was continued for a total of 7 h following embolization. Complete clot lysis was demonstrated in 6 of 7 animals. Brain infarct size (triphenyltetrazolium chloride staining) was 36.0 +/- 12.9% hemisphere (mean +/- SEM). Both clot lysis rate and infarct size were very similar to that previously seen following administration of t-PA alone (58% and 31.6 +/- 6.4% hemisphere, respectively) but in marked contrast to previous results seen with intravenous aspirin (no clot lysis). These results suggest that antiplatelet agents used clinically for stroke prophylaxis (aspirin or ticlopidine) may influence the success rate of thrombolysis following initiation of thrombolytic therapy for acute thromboembolic stroke.

Structure-based design of lipophilic quinazoline inhibitors of thymidylate synthase.

To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.

"Birds of a feather...": behavioral concordances and preferential personal attraction in children.

The purpose of this study was to assess the extent to which preferential personal attraction was associated with behavioral similarity among newly acquainted children. Participants included 69 focal children, selected from a sample of 236 7-year-old children who met, for the first time, in same-sex quartets (n = 59) for a free-play session. Within each of these quartets, a "discriminating child" was identified; this child expressed a clear preference for one of his or her playmates over one other of her or his quartet playmates. Preference was determined sociometrically after the children became acquainted during free play. The results indicated that "discriminating" children were more behaviorally similar to preferred playmates than to nonpreferred playmates both in terms of cognitive play style and social participation. Implications of the findings are discussed in terms of the relation to the acquaintanceship process.

Attending behavior: a descriptive study of children aged 18 through 23 months.

The purpose of this study was to provide descriptive data on the attending behavior of children aged 18 through 23 months. The method used was designed to be clinically feasible for occupational therapy practitioners. Forty-eight children between the ages of 18 and 24 months were studied through observation of a 15-min session of free play with a standard set of toys. The child's physical contact with objects was used as a guideline for timing. Four factors were examined: total time attending, total number of activities attended to, average attending time per activity, and longest time attending to one activity. The Mann Whitney U statistic revealed that there were no significant differences between boys and girls on any of the four factors. There were, however, statistically significant differences between children aged 18 through 20 months and children aged 21 through 23 months for all of the factors except longest time attending to one activity. The children in this study changed activities frequently (median of 10 activities in 15 min) and attended briefly (median attending time per activity of 81 sec). They were, however, capable of attending to one activity for much longer periods of time (median of 225 sec). With minimal adult intervention, the children spent an average of 12.5 min attending to the toys during a 15-min session. These results may augment other aspects of occupational therapy assessment by offering some insight into whether or not an individual child demonstrates age-appropriate attending behavior. Additionally, this standardized method of observation may offer occupational therapists a clinically feasible means of assessing attending behavior.